Piezoelectric microcantilever sensors for biosensing

ABSTRACT

A piezoelectric microcantilever for sensing compounds or molecules. The piezoelectric microcantilever, may include at least one electrode, an insulation layer, a receptor, an immobilization layer, a non-piezoelectric layer and a piezoelectric layer The sensor is capable of self actuation and detection. The piezoelectric layer may be constructed from a highly piezoelectric thin lead magnesium niobate-lead titanate film, a highly piezoelectric thin zirconate titanate film, a highly piezoelectric lead-free film. Methods of using the sensors and flow cells and arrays including the sensors are also described.

This application is a divisional of U.S. patent application Ser. No.12/514,941 filed Nov. 18, 2009, which is a 371 of International PatentApplication No. PCT/US2007/085771, filed Nov. 28, 2007, which in turnclaims the benefit of U.S. Provisional Patent Application No. 60/867,538filed Nov. 28, 2006.

STATEMENT OF GOVERNMENT INTEREST

This invention was reduced to practice with Government support underGrant No. R01 EB000720 awarded by the National Institutes of Health; theGovernment is therefore entitled to certain rights to this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to piezoelectric microcantilever sensors forbiosensing. More specifically, the invention relates to highly sensitivepiezoelectric microcantilevers capable of determining the presenceand/or mass of organic compounds. Applicable fields of use may includebiodefense, food safety and pathogen detection.

2. Description of the Related Technology

Current biosensing technologies utilize quartz crystal microbalances(QCM), silicon microcantilevers, electrochemical enzyme immunoassays,fluorescence, laser-based or fiber-optics-based methods, amplificationschemes such as polymerase chain reaction (PCR), or bound metalparticles to determine the presence and/or mass of organic compounds.These techniques, however, fail to provide quantitative, efficient orhighly sensitive detection. In addition to lacking sensitivity, they arealso incapable, in some cases, of simultaneously monitoring multiplecompounds or being used in high throughput array applications.

TABLE 1 Comparison of PEMS with Other Biosensors Detection Label-Direct, High- Detector Type sensitivity free In-situ Rapid throughputMultiplexing PEMS 10⁻¹⁵-10⁻¹⁸ g yes yes yes yes yes QCM 10⁻⁹ g yes yesYes No no Silicon 10⁻¹² g yes no No No no microcantilever Optical fiberno no No Yes yes fluorescence ELISA²⁶ 10⁻¹⁰ g no no No Yes yes SPR³10⁻¹² g yes yes Yes No no

Of these technologies, QCM, which utilizes thickness-mode resonancesensing, is one of the most common commercially available biosensingtechnologies. Detection sensitivity of a QCM is related to the resonancefrequency and the thickness of the quartz membrane. A resonancefrequency of about 5 MHz, corresponding to a quartz membrane thicknessof 330 μm, enables a minimum detectable mass density of about 10⁻⁹g/cm². Sensitivity is therefore generally limited to a range of about10⁻⁸ g/Hz.

To increase sensitivity, some biosensors utilize silicon-basedmicrocantilevers, which offer a sensitivity of approximately 10⁻¹² g/Hz,about three orders of magnitude higher than QCMs. Advantageously,silicon microcantilevers are also widely available and may be easilyintegrated with existing silicon fabrication methodologies. Most siliconmicrocantilevers, however, rely on complex external optical componentsfor deflection detection, an external driving mechanism for actuationand also require laser alignment. Moreover, because they are notpiezoelectric, silicon microcantilevers are inferior for in-solutionsensing, yielding low resonance peaks upon immersion in a solution.

Piezoelectric cantilevers, in comparison, use electrical means fordetection and are not encumbered by the complexity and mass of thesilicon-based sensors. Constructed from lead zirconate titanate (PZT),they are capable of electrical self-excitation and self-sensing forin-situ electrical detection. Currently, piezoelectric biosensors aremillimeter-size cantilevers made by bonding commercial PZT tonon-piezoelectric substrates such as stainless steel, titanium or glass.Although capable of in-situ biosensing, these millimeter-sizecantilevers lack the desired sensitivity for such applications.

Thin-film-based PZT microcantilevers, such as those disclosed inJP-07027559 A2 and U.S. Pat. No. 7,084,554, are highly sensitiveinstruments. U.S. Pat. No. 7,084,554, in particular, discloses a thinpiezoelectric film biomorph capable of being formed as a cantilever. Thebiomorph may be composed of a piezoelectric thin PZT film of about 1-10μm in thickness for the purpose of increasing the working frequencyrange of micro-electro-mechanical dimensioned (MEMS) systems. The patentfurther teaches that the piezoelectric thin film may be fabricated bythin film fabrication methods such as a sol-gel method, sputtering,hydrothermal methods, chemical vapor deposition (CVD) or another thinfilm fabrication method, followed by low temperature annealing and dryetching, plasma etching or patterning by wet chemical etching (See col.5, lines 55-64 of U.S. Pat. No. 7,084,554). These piezoelectricmicrocantilevers, however, are incapable of in-situ electrical detectiondue to degradation of resonance peaks in solution.

Recent advances in thin-film PZT microcantilevers incorporate anelectrical insulation layer that prevents liquid damping. U.S. PatentPublication no. 2005/0112621 discloses an insulation layer surrounding aPZT microcantilever having a thin piezoelectric film in order to preventconduction in liquid media (See e.g. col. 4, lines 28-36).

However, there remains a need for a piezoelectric microcantilever devicecapable of in-solution biologic detection having a femtogram or highersensitivity. In addition, there remains a need for a device consistingof array piezoelectric microcantilevers that is capable of simultaneousdetection of multiple compounds.

Advancement in the sensitivity, accuracy and efficiency of electricalbiosensing is essential to the developing field of bioterrorism defense.During the fall of 2001, for example, bacillus Anthracis spores, abioterrorism agent, were responsible for the deaths of 5 individuals andinfection of 17 others. As bioterrorism threats become more prevalent,there is a growing need for reliable in-situ detectors capable ofefficiently detecting multiple biological agents in real time.

Advancements in biosensing accuracy, sensitivity, multi compounddetection are also potentially useful in the health sciences for earlydetection and prevention of diseases. Breast cancer, for example, is thesecond leading cause of death for women. Although a number of potentialbreast cancer markers, such as HER2 (HER2/neu, c-erbB-2), EGFR, CA-15-3,CA27.29, urokinase plasminogen activator receptor (uPAR),carcinoembryonic antigen (CEA), α-fetoprotein (AFP), and cytokeratins,are known, no blood test or method for detecting these markers currentlyexists. Mammography is frequently inadequate and often produces falsepositives leading to unnecessary biopsies. Therefore early detectionmethods for various cancers and other diseases lacking adequatediagnostic means, like breast cancer, that are capable of accurately,effectively and non-invasively identifying and quantifying pathogens andother disease markers are also needed.

SUMMARY OF THE INVENTION

In a first aspect, the present invention is directed to piezoelectricmicrocantilever sensors useful for detecting organic compounds. Thepiezoelectric microcantilever includes a piezoelectric layer, anon-piezoelectric layer and a receptor. The microcantilever may beconstructed from piezoelectric lead magnesium niobate-lead titanate(Pb(Mg_(1/3)Nb_(2/3))O₃)_(0.65)—(PbTiO₃)_(0.35)(PMN_(0.65)—PT_(0.35))(PMN—PT) which typically has a thickness notgreater than 70 μm with a dielectric constant of at least 1600.

In a second embodiment, the piezoelectric layer of the microcantileveris constructed from lead zirconate titanate (PZT), which typically has athickness not greater than 4 μm with a dielectric constant of at least1600.

In another aspect, the present invention is directed to methods forbioterrorism defense, food safety and pathogen detection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross section of one embodiment of a piezoelectricmicrocantilever in accordance with the present invention.

FIG. 2( a) is a graph of piezoelectric coefficient, −d₃₁, versuselectric field for PMN microcantilevers in accordance with the presentinvention.

FIG. 2( b) is the resonance spectrum of a PMN—PT PEMS

FIG. 3 is a schematic showing a preferred micro-fabrication procedurefor constructing PZT/SiO₂ microcantilevers in accordance with thepresent invention.

FIG. 4( a) is a graph of quality value (Q) versus cantilever length forPZT cantilevers in accordance with the present invention.

FIG. 4( b) is a graph of quality value (Q) versus cantilever length forPZT and PMN—PT PEMS in accordance with the present invention.

FIG. 5( a) depicts a flow cell system which can be used in conjunctionwith the cantilevers of the present invention.

FIG. 5( b) depicts a 3.5 in by 7.5 in portable PEMS sensor capable ofworking with 8 sensors and powered by a 9-V battery.

FIG. 6 is a graph of Δm/Δf as a function of cantilever length.

FIG. 7 is a graph of resonance frequency shift as a function of time forthe microcantilever PEMS-B.

FIG. 8 is a graph of resonance frequency shift as a function of time forthe microcantilever PEMS-B using a resonance peak of 285 kHz.

FIG. 9 is a graph of resonance frequency shift as a function of time forthe microcantilever PEMS-A.

FIG. 10 is a graph of resonance frequency shift as a function of thetotal number of BA spores for both microcantilevers PEMS-A and PEMS-B.

FIG. 11( a) depicts an experimental setup for determining the resonancefrequency shift for a 1 mm long PZT/glass cantilever having a 2 mm longglass tip.

FIG. 11( b) is a graph of resonance frequency shift as a function oftime of the PEMS of FIG. 11( a).

FIG. 12 is a graph of frequency shift as a function of PSA concentrationfor various cantilevers

FIG. 13( a) is a graph of resonance frequency shifts of threepiezoelectric cantilevers as a function of time in a HER2 solution.

FIG. 13( b) is a graph of resonance frequency shifts of threepiezoelectric cantilevers as a function of time in a PSA solution.

FIG. 13( c) is a graph of resonance frequency shifts of threepiezoelectric cantilevers as a function of time in a gp120 solution.

FIG. 14( a) shows the resonance spectra of a parylene insulated PEMSplaced in static and 1 ml/min flow solutions of PBS.

FIG. 14( b) shows graphs of peak shift as a function of time for aparylene insulated PEMS placed in static and 1 ml/min flow solutions ofPBS.

FIGS. 15( a)-15(b) are resonance spectra of a MTMS insulated PZT PEMSplaced in a 0.5 ml/min flow solution of PBS.

FIG. 16( a) shows a schematic diagram of binding an NHS ester of theSMCC to a primary amine of the scFv.

FIG. 16( b) shows the formation of a thioether bond between themaleimide of the SMCC and the sulfhydryl group of the MPS on the sensorsurface.

FIG. 17( a) shows a graph of resonance frequency as a function of timeduring scFv immobilization, PBS rinsing, blocking in a 10 mg/ml of BSAsolution, and detection in a 100 ng/ml pure HER2 solution for aPMN—PT/Sn PEMS insulated by MPS.

FIG. 17( b) shows resonance spectra at various time frames of FIG. 17(a).

FIG. 17( c) shows a graph of resonance frequency as a function of timeduring scFv immobilization, PBS rinsing, blocking in a 10 mg/ml of BSAsolution, and detection in a 100 ng/ml of HER2 solution and 1 mg/ml BSAsolution for a PMN—PT/Sn PEMS insulated by MPS.

FIG. 18 is a graph of resonance frequency shift as a function of time ofa PMN—PT/Sn PEMS insulated with MPS with scFv immobilized via SMCCcoupling at 5, 10, and 50 ng/ml of HER2. The insert is a blow-up thatshows the resonance frequency shift at 5 and 1 ng/ml HER2.

FIG. 19 is a graph of resonance frequency as a function of time of aone-sided PMN—PT/tin PEMS during the various stages of detection of HER2at 1 ng/ml in a background of 1 mg/ml BSA.

FIG. 20 is a graph of resonance frequency as a function of time showingthe detection of HER2 in solutions containing diluted 1:40 fetal bovineserum and HER2 in a concentration of about 110 pg/ml, 50 pg/ml, 30 pg/mlor 10 pg/ml using a PMN—PT/Cu PEMS.

FIG. 21( a) is a graph of resonance frequency shift as a function oftime showing the detection of HER2 in a solution of diluted (1:40) fetalbovine serum containing a 6 ng/ml concentration of HER2 using threedifferent receptors.

FIG. 21( b) is a graph of resonance frequency shift as a function oftime showing the detection of HER2 in a solution of diluted (1:40) fetalbovine serum containing a 60 ng/ml concentration of HER2 using threedifferent receptors.

FIG. 21( c) is a graph of resonance frequency shift as a function oftime showing the detection of HER2 in a solution of diluted (1:40) fetalbovine serum containing a 600 ng/ml concentration of HER2 using threedifferent receptors.

FIG. 22( a) is an optical micrograph of the PZT/glass PEMS array used inthe CP detection and

FIG. 22( b) is a graph of resonance frequency shift as a function oftime showing the detection of CP at a concentration of about 1 CP/mlusing 4 different PEMS.

FIG. 23( a) is an SEM micrograph a 40 μm long PZT/SiO₂ PEMS.

FIG. 23( b) is a graph of resonance frequency shift versus relativehumidity of a 40 μm long PZT/SiO₂ PEMS.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Piezoelectric microcantilever sensors (PEMS) are mass sensors that usean electrical means for detection. Receptors are coated on the surfaceof PEMS to bind molecules of interest. The PEMS detects a change in massbecause newly bound target molecules shift the mechanical resonancefrequency of the device. By monitoring the resonance frequency shifts, aPEMS is capable of rapid, label-free, in situ quantitative detection oforganic compounds or molecules including pathogens, antigens andproteins in a small volume solution (e.g. 100 μl) or in a cell cultureusing simple all-electrical measurements. PEMS are capable of electricactuation and detection and may also be constructed as an array forsimultaneous monitoring of multiple target compounds or molecules.

FIG. 1 shows the basic structure of a microcantilever sensor. A PEMSincludes a conductive element 1 and a second conductive element 2(bottom electrode), electrically insulating layer 3, receptorimmobilization layer 4, receptors 5, at least one non-piezoelectriclayer 6, and at least one piezoelectric layer 7. The PEMS shown in FIG.1 also includes electrical leads 9.

Conductive elements 1, 2 may be any element capable of conducting anelectrical signal from the piezoelectric layer to a device for detectingthat signal. In a preferred embodiment, conductive elements 1 and 2 areelectrodes which may be constructed from any conductive material.Preferably, the first electrode 1 is constructed from Au/Cr or Pt/Ti andsubsequently patterned in several regions. The second electrode 2 ispreferably constructed from Pt/TiO₂ on SiO₂ for PZT/SiO₂ PEMS or Pt/Tion a metal substrate or non-piezoelectric layer and subsequentlypatterned as well.

In order to maintain functionality in solution by preventing conduction,it may be useful to electrically separate or buffer conductive element 1and second conductive element 2. Conductive element 1 is patternedslightly smaller than the piezoelectric layer 7 to ensure completeinsulation of the edges and corners thereof. Any electrically insulatinglayer 3 may be used as a coating to achieve electrical separation orbuffering.

In one embodiment, insulating layer 3 may comprise a 1.5 μm thickparylene (poly-para-xylylene) coating deposited on an electrode bychemical vapor deposition. When placed in static and 1 ml/min flow rateof PBS solution, a parylene insulating layer 3 essentially preventsbackground resonance frequency shifts greater than 30 Hz and 60 Hz,respectively, over a period of 30 minutes. As a result, insulating layer3 enables complete submersion of the microcantilever for in situ orin-solution detection while maintaining a Q value (quality value)greater than 35. For the purposes of this patent application, Q value isdefined as the ratio of the resonance frequency to the resonance peakwidth at half the peak height.

Alternatively, a PEMS may be insulated using self-assembled monolayerswith hydrophobic properties, preferably methyltrimethoxysilane (MTMS) ora combination of MTMS with parylene coatings of varying thicknesses, mayalso be used. When immersed in a PBS solution, an MTMS insulatedpiezoelectric microcantilever yields strong resonance peak intensitiesand prevents background resonance frequency shifts greater than 30 Hzover a period of 30 minutes.

Other insulation materials may include 3-mercaptopropyltrimethoxysilane, Al₂O₃, SiO₂ and any functional hydrophobic silane,having a hydrophobic group selected from the group consisting of alkyl,phenyl, alkyl halide, alkene, alkyne, and sulfhydryl. In a preferredembodiment, the insulation material is mercaptopropylsilane (MPTS),which can also function to immobilize a receptor on the cantilever. Theinsulating materials may also include any combination of any of MTMS,MPTS, parylene, 3-mercaptopropyl trimethoxysilane, Al₂O₃, SiO₂, anyfunctional hydrophobic silane having a hydrophobic group selected fromthe group consisting of alkyl, phenyl, alkyl halide, alkene, alkyne, andsulfhydryl, or a combination thereof.

Receptors 5 may be densely packed and immobilized onto, for example, abi-functional linker modified sensor surface. Any receptor, such asspecially synthesized cavitants, DNA oligonucleotides, proteins, singlechain variable fragments (scFvs), enzymes, and antibodies to cells,antigens, pathogens, viruses, parasites, or combinations thereof may bebound to the sensor surface. For example, when trying to detect tumors,monomeric and dimeric anti-tumor scFv molecules, which are composed ofvariable light and heavy chains of antibody molecule anti-ECD scFV, thatreact to cancer markers may be bound to the electrodes. Similarly, whentrying to detect Bacillus anthracis (“BA”), antibodies specific to BAspore surface antigens may be immobilized on the electrodes.

Any means of adhering receptors 5 to the sensor surface may be utilized.In a preferred embodiment, receptors 5 are bound to the electrodes usingan immobilization coating 4, such as self assembled monolayers (“SAM”),MPTS and bi-functional linkers. In one embodiment, for purposes ofbinding scFv, the immobilization coating may be a self assembledmonolayer of 3-mercaptoproprionic acid (MPA) on a copper and gold-coatedelectrode activated with 1-ethyl-3-(3-dimethylaminopropy)carbodimidehydrochloride (EDC) and 5 mg/ml N-hydroxysulfosuccinimide (NHS).

The PEMS also includes at least one non-piezoelectric layer 6, which maybe fabricated from any compatible material, including a ceramicmaterial, a polymeric material, a metallic material or combinationsthereof. Preferably the non-piezoelectric layer 6 is fabricated fromsilicon dioxide (SiO₂) and silicon nitride (Si₃N₄) for PZT-thin filmbased PEMS. For example, a silicon nitride coating on single crystalsilicon wafer may be prepared by low press chemical vapor deposition. Alow stress silicon dioxide layer may subsequently be deposited on thesilicon nitride layer by growing silicon dioxide films using lowtemperature oxide deposition or plasma enhanced chemical vapordeposition. For PMN—PT-based PEMS, the non-piezoelectric layer can beany ceramic, metallic, or polymeric layer. A metallic layer such as Cu,tin, Ni, Ti, etc., or any combination is preferred because it can beprovided by simple electroplating.

Non-piezoelectric layer 6 may be bonded to a shorter piezoelectric layer7 so that the portion of non-piezoelectric layer 6 extending beyond theend of piezoelectric layer 7 forms a non-piezoelectric tip. Bothpiezoelectric layer 7 and non-piezoelectric layer 6 may be attached to aclamp. In an alternative embodiment, piezoelectric layer 7 may extendbeyond non-piezoelectric layer 6, forming a piezoelectric tip.Optionally, the PEMS may be constructed so that neither piezoelectriclayer 7 nor the non-piezoelectric layer 6 extends beyond the other. Inorder to achieve the best results, one of the piezoelectric 7 andnon-piezoelectric layers 6 preferably extends beyond the other to form atip. A PEMS may also include multiple piezoelectric andnon-piezoelectric layers. For example, a non-piezoelectric layer may beplaced between two piezoelectric layers or a piezoelectric layer may beplaced between two non-piezoelectric layers.

A significant aspect of the microcantilever device is the fabrication ofa highly piezoelectric layer 7, which enables electrical detection andactuation within the cantilever. The piezoelectric layer may function asa driving element, vibrating element and sensing element. Applying an ACvoltage (input) across the piezoelectric layer bends and vibrates thePEMS, which in turn induces a piezoelectric voltage that producesreadily detectable changes in the magnitude and phase of the outputvoltage. The resonance frequency of the PEMS is obtained by monitoringthe maximum of the phase shift of the output voltage. This measurementis accomplished all-electrically, i.e., electrical actuation andelectrical sensing.

Piezoelectric layer 7 may be constructed from any piezoelectricmaterial, including a lead-free piezoelectric material such as(Na_(0.5)K_(0.5))_(0.945)Li_(0.055)Nb_(0.96)Sb_(0.04)O₃ (hereinafter“Sb—NKNLN”), Sb—(Na_(0.5)K_(0.5))NbO₃—LiTaO₃ (hereinafter “Sb—NKNLT”),Sr—(Na_(0.5)K_(0.5))NbO₃—LiTaO₃ (Sr—NKNLN),Sr—Na_(0.5)K_(0.5))NbO₃—LiTaO₃ (Sr—NKNLT),SbSr—(Na_(0.5)K_(0.5))NbO₃—LiTaO₃ (SrSb—NKNLN),SrSb—Na_(0.5)K_(0.5))NbO₃—LiTaO₃ (SbSr—NKNLT), solid solutions with(Bi_(0.5)K_(0.5))TiO₃, (Bi_(0.5)Na_(0.5))TiO₃, Ba(Zr_(x)Ti_(1-x))O₃,BaTiO₃ (hereinafter “BT”), (Bi_(1/2)K_(1/2))TiO₃ (hereinafter “BKT”),(Bi_(1/2)Na_(1/2))TiO₃ (hereinafter “BNT”), Ba(Zr_(x)Ti_(1-x))O₃(hereinafter “BZT”), Bi(Zn_(1/2)Ti_(1/2))O₃ (hereinafter “BiZT),(Na_(x)K_(1-x))NbO₃ (hereinafter “NKN”), BiScO₃—PbTiO₃BaTiO₃—(Bi_(1/2)K_(1/2))TiO₃ (hereinafter “BKBT”),(Bi_(1/2)Na_(1/2))TiO₃—(Bi_(1/2)K_(1/2))TiO₃ (hereinafter “BNKT”),(Bi_(1/2)Na_(1/2))TiO₃—BaTiO₃ (hereinafter “BNBT”),(Bi_(1/2)Na_(1/2))TiO₃—Ba(Zr_(x)Ti_(1-x))O₃ (hereinafter “BNBZT”) and(Bi_(1/2)Na_(1/2))TiO₃—BaTiO₃—(Bi_(1/2)K_(1/2))TiO₃ (hereinafter“BNBK”).

In a preferred embodiment, the piezoelectric layer is fabricated fromhighly piezoelectric lead magnesium niobate-lead titanate films, e.g.(Pb(Mg_(1/3)Nb_(2/3))O₃)_(0.65)—(PbTiO₃)_(0.35)(PMN_(0.65)—PT_(0.35))(PMN—PT), highly piezoelectric lead zirconatetitanate (PZT) films or high piezoelectric lead-free films.Additionally, piezoelectric layer 7 may be fabricated in any form,preferably having free standing film geometry to enhance domain wallmotion and piezoelectric performance.

A piezoelectric PMN—PT layer may be fabricated using aprecursor-suspension method. Submicron crystalline PMN powder is firstprepared by dispersing Mg(OH)₂-coated Nb₂O₅ particles in a leadacetate/ethylene glycol solution followed by calcination at about 800°C. The crystalline PMN powder was subsequently suspended in a leadtitanate (PT) precursor solution containing lead acetate and titaniumisopropoxide in ethylene glycol to form a PMN—PT precursor powder, whichcan be sintered at a temperature as low as about 900° C.

The resulting suspensions have previously been used to formulate PMN—PTfreestanding films of about 8-75 μm thick upon sintering at 1000° C.Preferably, the thickness is about 1 μm-127 μm, more preferably lessthan about 50 μm, and most preferably less than about 8 μm. Theprecursor-suspension method may also be used to produce freestandingPMN—PT films having a thickness of less than 8 μm or larger than 75 μm.Typically, the freestanding PMN—PT films have femtogram sensitivity ofat least 2×10⁻¹⁴ g/Hz, dielectric constants greater than 1000, saturatedpolarization of about 30 μC/cm², remnant polarization of 25 μC/cm² and aQ value as high as 300 and as low as 20. Therefore PMN—PTmicrocantilevers are capable of generating higher-mode resonance peaksresulting in enhanced sensitivity detection.

Further, PMN—PT freestanding films with thicknesses of 8 μm or 22 μmwere found to exhibit an extremely high electric-field enhancedpiezoelectric −d₃₁ coefficient of 2000 μm/V at E=10 kV/cm, as shown inFIG. 2( a), which is about 7 times that of the bulk PMN—PT andcommercial PZT, and which also exceeds the piezoelectric coefficient ofspecially-cut single crystalline PMN—PT. The direction of the trianglesin FIG. 2( a) indicate whether the results were obtained when the fieldwas ramped up or down. The piezoelectric coefficient −d₃₁ is calculatedaccording to (Equation 1),

$\begin{matrix}{{d_{31} = {\frac{h_{can}}{3{VL}^{2}} \cdot \frac{{E_{1}^{2}t_{1}^{4}} + {E_{2}^{2}t_{2}^{4}} + {2E_{1}t_{1}E_{2}{t_{2}\left( {{2t_{1}^{2}} + {2t_{2}^{2}} + {3t_{1}t_{2}}} \right)}}}{E_{1}E_{2}{t_{1}\left( {t_{1} + t_{2}} \right)}\left( {1 - v} \right)}}},} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$where V is the applied voltage, L, the cantilever length, v the Poissonratio, E₁, and E₂ (t₁ and t₂) are the Young's modulus (thickness) ofcopper and that of PMN—PT, respectively. The large piezoelectriccoefficient −d₃₁ is believed to be due to (1) low-temperature sinterablePMN—PT powder that enables the film to sinter below 1000° C. to avoidlead loss and maintain the correct stoichiometry, and (2) freestandingfilm geometry that aligns the polarization in the plane of the film.Polarization reorientation by the applied electric field, normal to thefilm, enhances the piezoelectric coefficient. By using sinterable PMNpowders and eliminating defects through careful placement of the thinfilms on a substrate, the PMN—PT films were endowed with superiorpiezoelectric properties. This fabrication method is robust and may bereadily applied to other lead based piezoelectrics and non-lead-basedmaterials.

In another embodiment a PEMS may be constructed from highlypiezoelectric lead zirconate titanate (PZT). (PZT)/SiO₂ PEMS may beconfigured to form microcantilevers less than 20 μm long having attogramsensitivity of 10⁻¹⁸ g/Hz, a piezoelectric layer thickness of about1.0-μm-8 μm, more preferably, less than about 4 μm, about 1.5-μm-2 μm orless than about 2 μm and a dielectric constant of at least 1600, morepreferably, at least 1900. The PZT PEMS is capable of exhibiting up tofour resonance peaks with quality values, Q, ranging from a minimum ofabout 20 to between 120 and 320. (PZT)/SiO₂ PEMS may also be configuredto have a thickness of less than 2 μm and a length of less than 30 μm.The dielectric constants can be further raised to above 2000 and up toabout 3000 by any standard method of doping the piezoelectric layer.

A PZT/SiO₂ piezoelectric layer 7 of film may be formed on silicon wafersand attached to a substrate, such as glass, to form an array. It is alsopossible to fabricate thin PZT films, of 1.0 to 2.0 μm in thickness, ona variety of different substrates using a sol-gel process. FIG. 3depicts a preferred micro-fabrication procedure for constructingPZT/SiO₂ piezoelectric microcantilever sensors.

One advantage of this invention is the ability to construct amicrocantilever from one piece of PZT/SiO₂ film without having toseparately attach the non-piezoelectric 6 and piezoelectric layers 7.Because the cantilevers thus fabricated have clean interfaces betweenthe PZT/SiO₂ layer and the electrodes, the cantilevers exhibit high Qvalues. The resultant clean geometry of the microcantilever increasessensitivity and facilitates the manufacturing process.

The self-actuating and self-detecting PEMS of the present inventionovercomes a significant problem of the prior art, miniaturizingmicrocantilevers without losing high resonance-peaks. In the presentinvention, the novel PMN and PZT micro-fabrication methods produce thinpiezoelectric PEMS having enhanced sensitivity, as evidenced by thelarge dielectric constants, piezoelectric coefficients and Q values.

Q values are an excellent indicator of cantilever sensitivity. Althoughit is expected that the Q value of silicon microcantilevers decreaseswith cantilever size, FIG. 4 shows that the Q value of acommercial-PZT-based piezoelectric microcantilever remains approximatelythe same even though the microcantilever length varies over two ordersof magnitude due to the polycrystalline composition of the piezoelectriclayer 7. The piezoelectric layer 7 of the present invention thereforeutilizes polycrystalline compositions such as PMN—PT and PZT in order tominiaturize PEMS without losing sensitivity. For example, by reducingPZT PEMS to a length-scale of 300 μm and 50 PMN—PT and PZT PEMS arecapable of attaining detection sensitivity greater than 10⁻¹⁵ and 10⁻¹⁸g/Hz, respectively. Freestanding PMN—PT PEMS 500-700 μm in length arecapable of achieving Q values higher than 300. Additionally, PEMS with athickness of less than 5 μm and length of less 120 μm long, may achievea sensitivity better than 10⁻¹⁶ g/Hz.

To further increase sensitivity and expedite the detection process, thePEMS may be immersed in a flowing solution for in-solution detection.The PEMS is preferably situated in a flow cell system to enabletailored, rapid and simultaneous detection and quantification ofmultiple organic compounds or molecules.

FIG. 5( a) shows a flow cell system 10, with a PEMS holder/measuringunit 11, having a total volume of less than 0.03 ml, pump 12, and amechanism for controlling temperature and humidity (not shown). The flowcell 10 may attain flow rates of up to 1 ml/min. The total volume of theflow cell, number of channels and flow rate may vary depending upon thenumber of compounds to be measured. The flow cell 10 may cooperate witha portable PEMS unit, shown in FIG. 5( b), which has multiple channelsfor the simultaneous quantification of multiple receptor specificmolecules. The portable PEMS is inexpensive and capable of obtainingquick measurements.

Another means for further enhancing sensitivity is by increasinghumidity. The mass change per unit area per percent humidity change ofPZT PEMS is estimated to be about 1.2×10⁻¹¹ g/Hz/mm²/% humidity. Thesensitivity of PMN PEMS by comparison is known to be about three timesgreater than that of PZT PEMS.

The resultant PEMS are chemically inert, thermally stable andminiaturized to enhance sensitivity. They function by binding targetmolecules that react to the receptors immobilized on the electrodes. Thecorresponding change in mass shifts the mechanical resonance frequencyof the microcantilever. The PEMS is capable of detecting these shifts inresonance frequency by monitoring the i^(th)-mode flexural resonancefrequency f_(i), which is related to the effective spring constant,K_(e), and effective mass, M_(e), of the piezoelectric cantilever at thetip as shown in Equation 2.

$\begin{matrix}{f_{i} = {\frac{1}{2\pi}\sqrt{K_{e}/M_{e}}}} & \left( {{Equation}\mspace{14mu} 2} \right)\end{matrix}$The binding of a receptor specific molecule to the cantilever surfacechanges the cantilever mass and the cantilever spring constant. Theresonance frequency shift Δf, expressed in Equation 3,

$\begin{matrix}{{{\Delta\; f_{i}} = {f_{i}\left( {{- \frac{\Delta\; m}{2M_{e}}} + \frac{\Delta\; k}{2K_{e}}} \right)}},} & \left( {{Equation}\mspace{14mu} 3} \right)\end{matrix}$where Δm and Δk denote the mass change and the effective springconstant, model the functionality of the microcantilever. FIG. 6 showsthe Δm/Δf of PMN—PT/Cu PEMS constructed from 8 μm and 22 μm thick PMN—PTfilms. The full circles and full squares of FIG. 6 represent resultsobtained with millimeter long PEMS, with and without a stainless steeltip, respectively. The Δm/Δf of these PEMS deviates from the solid lineby more than three orders of magnitude, thereby indicating that reducingthe PEMS thickness and dimension binding-related stress greatly enhancesthe resonance frequency shift.

These PEMS may be used for various sensing applications such assolid-liquid transition detectors, liquid viscosity and density sensors,mass sensors for in situ and in-water detection. PEMS may generally beused for detection of any molecule or organic compound.

The PEMS technology may be particularly promising for the detection ofbioterrorism agents. Antibody receptors specific to at least onebioterrorism agent may be bound to an electrode and used to detect thepresence of a bioterrorism antigen. In addition to identifying theexistence of a bioterrorism agent, it may also be used to quantify theconcentration of the agent.

Additionally, PEMS may be useful in the health sciences as a diagnosticinstrument. It may be used as a means for early detection of cancers andother diseases. It may also be used to monitor the progress of thedisease throughout treatment. The PEMS may be incorporated in a portabledevice and used as a noninvasive means for testing blood and otherbodily fluids for various pathogens, infectious agents and other markersindicative of disease.

PEMS may also be particularly applicable for the food science and foodmanufacturing industry. PEMS may be used as a diagnostic instrument fordetecting pathogens or other disease agents present in food supplies andprepared or processed foods. Additionally, it may also be useful inmanufacturing plants and food service industries as a means ofintermittently checking food products during different phases of foodpreparations thereby preventing contamination and the spread ofbacterial or viral diseases such as salmonella and E. coli.

EXAMPLES Example 1

PMN—PT/Sn PEMS were used in-situ to detect the presence of Bacillusanthracis (BA), a bioterrorism agent. Two PEMS, PEMS-A (500 μm long, 800μm wide, with a 22 μm thick PMN—PT layer, a 20 μm thick tin layer and a1×10⁻¹² g/Hz mass detection sensitivity) and PEMS-B (350 μm long, 800 μmwide, with an 8 μm thick PMN—PT layer, a 6 μm thick tin layer and a3×10⁻¹³ g/Hz mass detection sensitivity) were inserted in a flow cellthrough which a solution containing BA was pumped.

The experiment demonstrates that in phosphate buffered saline (PBS)solution, the PEMS were capable of achieving Q values ranging from 50 to75. PEMS-A exhibited resonance frequency shifts of 2100±200, 1100±100and 700±100 Hz at concentrations of 20000, 2000, and 200 spores/ml or16000, 1600, and 160 total spores, respectively. PEMS-B exhibitedresonance frequency shifts of 2400±200, 1500±200, 500±150 and 200±100 Hzat concentrations of 20000, 2000, 100, and 45 spores/ml or 16000, 1600,80, and 36 total spores, respectively.

The PEMS were fabricated from PMN—PT freestanding films 22 μm and 8 μmin thickness and electrically insulated with methyltrimethoxysilane(MTMS) coatings on the tin surface. A 30 nm thick nickel layer with a15-30 nm thick chromium bonding layer was first deposited on one side ofthe PMN—PT freestanding film by evaporation using an E-gun evaporator asthe electrode. A tin non-piezoelectric layer was then electroplated onthe nickel surface at a rate of 0.5 μm/minute, using tin sulfate in aplating solution. A 150 nm thick platinum on a 10-nm thick titaniumlayer was evaporated on the other face of the film as the otherelectrode. The resultant PMN—PT/Sn bilayer was embedded in wax and cutinto the cantilever shape with a wire saw. Gold wires were attached tothe top and bottom electrodes using conductive glue for electricalconnection. The PMN—PT/Sn strips were finally glued to a glass slide ora plastic substrate with epoxy to form the microcantilever geometry.

To insulate the tin electrode of these PMN—PT/Sn PEMS, the PEMS werefirst soaked in a diluted (1:40 in water) piranha solution (one part of98% sulfuric acid with one part of 30% hydrogen peroxide) at 20° C. for2 mM. Methyltrimethoxysilane (MTMS) was then spin coated on the tinsurface at 2000 rpm and cross linked (pH=9.5 for 2 hr) twice to form twoMTMS coatings on the tin surface.

For antibody immobilization, the platinum electrode was cleaned with a1:40 diluted piranha solution for 2 min followed by soaking the PEMS ina 2 mM 3-mercaptopropionic acid (MPA) for 2 hr to form an MPA monolayeron the Pt surface. The carboxyl group of the MPA was then activated by asolution of 2 mM N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC)and 5 mM N-hydroxysuccinimide (NHS) in water to allow covalent bondingof primary amines on the anti-BA-spore antibody to the MPA on theplatinum surface.

Anti-BA IgG antibodies were then immobilized on the platinum electrodeof the PMN-PT layer by first mounting the insulated PEMS with activatedMPA in a specifically designed holder and vertically inserting the PEMSinto the center of a custom polycarbonate flow cell, 1 mm wide by 1 mmhigh by 10 mm long with its major faces parallel to the flow and capableof holding a liquid volume of 0.8 ml. A baseline of pure phosphatebuffered saline (PBS) solution was run through the flow cell for severalminutes before injecting a 600 nM antibody solution into the flow cellfor 40 min, after which the flow system was once again rinsed with purePBS. The flow rate used for antibody immobilization and subsequent BAbinding was 1 ml/min, which corresponded to a Reynolds number of 1.6 anda flow speed of 2.5 cm/s over the PEMS surface.

Subsequently, 0.8 ml of the desired concentration of ultraviolet(UV)-killed, Sterne strain 7702 BA spores were injected into the flowsystem and run for 30 minutes. BA spores were then release from thesensor surface in a mixture of glycine and HCl at pH of 2.5 for 7 min.The total volume of the liquid utilized was only 0.8 ml; thus, theactual number of spores available for detection by the cantilever isactually only 80% of the indicated concentration. Scanning electronmicroscopy (SEM) was performed to confirm spore detection andsensitivity calculations.

FIG. 7 shows the time evolution of a 404 kHz resonance frequency ofPEMS-B during antibody immobilization, BA spore detection, and sporerelease. The antibody solution was introduced at t=0. The resonancefrequency shift during t=0-15 mM corresponds to the immobilization ofthe antibody to the PEMS platinum surface. The resonance frequencydecreased 0.4 kHz from 403.8 kHz at t=0 min to 403.4 kHz at t=15 min dueto the binding of the antibody to the MPA on the platinum surface. Thisresonance frequency shift saturated at t=10 min and Δf=−0.4 kHz,indicating the immobilization took about 10 min, approximately the sameas dipping in static solution. At t=15-20 min the antibody solution wasreplaced with PBS. As can be seen, there was no significant shift inthat period. At t=20 min, when 0.8 ml of a 20,000 spores/ml (16,000total spores) BA suspension was introduced into the flow cell, theresonance frequency rapidly decreased from 403.4 kHz at t=20 min to 401kHz at t=40 min with a Δf=−2.4 kHz. This resonance frequency shiftduring t=20-40 min corresponded to the binding of spores to the antibodyimmobilized on the PEMS surface. At t=60-65 min, the spore suspensionwas replaced by PBS, again resulting in no significant resonancefrequency shift in that period. From t=65-72 min, the release of thebound spores from the sensor surface in glycine/HCl solution wasperformed resulting in a recovery (up-shift) to 403.1 kHz at t=72 min,nearly the value before the detection. The second detection period with0.8 ml of a suspension of 20,000 spores/ml (16,000 total spores) wasstarted at t=72 min. As can be seen, the second detection at the sameconcentration exhibited a resonance frequency shift similar to the firstdetection. FIG. 7 therefore shows that the PEMS may be completelysubmerged in PBS while detecting the binding of proteins and spores tothe sensor surface by monitoring the resonance frequency down-shift of aparticular peak. Conversely the release of the antigen can be detectedby monitoring the resonance frequency up-shift as is shown during therelease of the spores by glycine/HCl solution.

FIG. 8 shows the plot of a resonance frequency shift of PEMS-B versustime using a resonance peak of 285 kHz at BA spore concentrations of20,000 spores/ml (16,000 total spores), 2000 spores/ml (1600 totalspores), 100 spores/ml (80 total spores), 45 spores/ml (36 total spores)and 12 spores/ml (10 total spores). Also shown inset in FIG. 8 areseveral in-PBS resonance spectra of PEMS-B during the BA sporesdetection at the 20,000 spores/ml concentration. The solid line, thedashed line, and the dashed-dotted line represent the resonance spectrumat t=0, 15 and 30 min, with Q values of 75, 65 and 69 respectively(where Q is ratio of the resonance frequency to the resonance peak widthat half of the peak height). As can be seen in FIG. 8, resonantfrequency shifts of 2400±200, 1500±200, 500±160, and 200±100 Hz wereobserved at t=30 min for 16000, 1600, 80, and 36 total spores,respectively. As the concentration was reduced to 12 spores/ml (10 totalspores), no discernable resonance frequency shift could be resolved fromthe sensor noise.

FIG. 9 shows a plot of the resonance frequency shift of PEMS-A versustime for concentrations of 20,000 spores/ml (16,000 total spores), 2,000spores/ml (1,600 total spores) and 200 spores/ml (160 total spores). Theinsert in FIG. 9 shows the in-PBS resonance spectra of PEMS-A at t=0,15, and 30 min with Q=62, 50, 53 as the solid, dashed, and dashed-dottedlines, respectively during the detection of BA spores at 16000 totalspores. As is shown in FIG. 9, at t=30 min these concentrations yieldedfrequency shifts of 2100±200, 1100±100, and 700±100 Hz, respectively.FIG. 4 also reveals that, for the lower spore levels of 160 and 1,600,the resonance frequency shift begins to level off at approximately 20minutes while for the higher spore level of 16,000, the resonancefrequency continues to decrease during the entire detection period assimilar to the situations with PEMS-B. The resonance frequency shiftsversus total number of BA spores of both PEMS-A and PEMS-B aresummarized in FIG. 10.

The experimentally obtained mass detection sensitivities are more than100 times more sensitive than the theoretical mass sensitivity valuessuggested by considering simply the mass loading effect alone.

Example 2

A PZT/SiO₂ piezoelectric cantilever sensor (PECS) 0.5 mm long and 2 mmwide, 5 MHz QCM and various PMN—PT PEMS were tested for in-situdetection of prostate specific antigen (PSA). The tip of each cantileverwere partially immersed in PSA solutions of 26.1, 17.5, 8.76, 4.38, and2.19 μg/ml to a depth of approximately 1 mm for approximately 30minutes. The tip was immersed in a vertical orientation in order toensure that there was no nonspecific binding due to gravity. Betweendetections, the antibody surface of each cantilever was regenerated by abrief dip in HCl-glycine solution.

The QCM sensor exhibited a resonance frequency shift of 45 Hz forantibody immobilization; PECS exhibited a resonance frequency shift of450 Hz. Using the QCM resonance frequency value and the Sauerbreyequation, the mass change per unit area due to the antibodyimmobilization was determined to be 3.5×10⁻⁹ g/mm. In comparison, thedetection sensitivity for a piezoelectric cantilever sensor, shown inFIG. 11( a), was determined to be approximately 3×10⁻¹¹ g/Hz. FIG. 11(b) and Table 2 shows the detection curves for the piezoelectriccantilever and demonstrates that the cantilever is only capable ofdetecting PSA at 4 μg/ml and above.

TABLE 2 Frequency shift at 20 minutes Detection Label- Direct, High-Detector Type sensitivity free In-situ Rapid throughput MultiplexingPEMS  10⁻¹⁵-10⁻¹⁸ g yes yes yes yes yes QCM 10⁻⁹ g yes yes Yes No noSilicon 10⁻¹² g yes no No No no microcantilever Optical fiber no no NoYes yes fluorescence ELISA²⁶ 10⁻¹⁰ g no no No Yes yes SPR³ 10⁻¹² g yesyes Yes No no

By comparison, the sensitivities of various PMN PEMS were determined bysubjecting the PEMS to a controlled 70% humidity and 16° C. environmentin which they were immersed in a flowing PSA solution of variousconcentrations. FIG. 12 shows that a 340 μm long, 750 μm wide and 8 μmthick PMN—PT/Cu microcantilever, represented in FIG. 12 by square datapoints, was found to have a sensitivity of 2×10⁻¹⁴ g/Hz; a 900 μm long,750 μm wide and 8 μm thick PMN—PT/Cu microcantilever, represented bytriangle data points in FIG. 12, was found to have a sensitivity 3×10⁻¹³g/Hz; and an 800 μm long, 750 μm wide and 22 μm thick PMN—PTmicrocantilever, represented by circle data points in FIG. 12, was foundto have a sensitivity of 5×10⁻¹³ g/Hz. Based on these sensitivities, itis possible to easily detect PSA at 3.5 ng/ml with a resonance frequencyover 500 Hz. Also plotted in FIG. 12 are the in situ resonance frequencyshifts obtained from the PZT/glass cantilever, which are represented bythe cross shaped data points. The intercept of the dashed lines with the50 Hz base line suggests the concentration limit of thesemicrocantilevers. For the in situ results of FIG. 12, the intercept wasdetermined to be the detection limit of the PZT/glass cantilever.

In situ detection is therefore predicted to have background resonancefrequency uncertainties of no more than 50 Hz. Using 50 Hz as a cutoffat the base line of FIG. 12, the extrapolated detection limit was 0.1ng/ml for microcantilever D with 2×10⁻¹⁴ g/Hz sensitivity, which wasalready as good as or better than the sensitivities of commercial PSAtesting techniques while the present in situ measurements have theadvantages of rapid detection and low cost.

Example 3

There exist a number of means for immobilizing various receptors onvarious different electrode surfaces. The immobilization methods maydiffer depending upon the receptor to be bound on the electrode surface.

For example, BA antigens may be immobilized on a lead surface bycleaning a platinum electrode with a 1:40 diluted piranha solution for 2min, soaking the PEMS in a 2 mM 3-mercaptopropionic acid (MPA) for 2 hrto form an MPA monolayer on the electrode surface, and activating thecarboxyl group of the MPA using a solution of 2 mMN-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC) and 5 mMN-hydroxysuccinimide (NHS) in water to allow covalent bonding of primaryamines on the anti-BA-spore antibody to the MPA monolayer.

A method for PSA receptor immobilization on a lead surface includes thesteps of immersing the microcantilever in a solution containing 1 part30% H₂O₂, concentrated H₂SO₄ and 29 parts water for 2 min, rinsing withdistilled water and immersing in a 1 mM MPA aqueous solution overnight.The microcantilever tip is then rinsed with distilled water andactivated by immersing in a solution containing 5 mg/ml1-ethyl-3-(3-dimethylaminopropy)carbodimide hydrochloride (EDC), 5 mg/mlN-hydroxysulfosuccinimide (NHS) and distilled water for 30 min. 1 μl ofa 0.1 mg/ml solution containing anti-PSA in PBS at a pH of 7.4 was thencoated on the cantilever surface for 30 min.

scFv receptors may be immobilized on a gold surface by binding activatedMPA to the amine of lysine in the scFv. The immobilization methodincludes the steps of coating MPA on the gold-coated cantilever tip,activating the MPA with a solution of 5 mg/ml1-ethyl-3-(3-dimethylaminopropy)carbodimide hydrochloride (EDC), 5 mg/mlN-hydroxysulfosuccinimide (NHS) and distilled water for 30 min andpartially immersing the microcantilever in the scFv solution.Alternatively, it may be possible to utilize longer mercaptoundecanoicacid (MUA) in order to optimize the efficiency.

scFv receptors may also be immobilized on a gold surface by using Cu²⁺ions to bind to His6 tag of scFv. In this method, the gold coatedmicrocantilever tip is first cleaned with Piranha solution for 10minutes and rinsed with distilled water. The gold surface is thentreated with MPTS, MPA (mercaptopropionic acid), or MUA(mercaptoundecanoic acid) to form a monolayer on the gold-coated sensorsurface. Preferably, the gold surface is treated by immersed in asolution of 1 mM 11 Mercaptoundecanoic Acid (MUA) and ethanol for 3hours and rinsed with distilled water. The cantilever is then immersedin a 2 mM Cu(ClO₄)₂ aqueous solution for 10 min to adsorb Cu²⁺ on theMUA self assembled monolayer and form a MPA-Cu composite monolayer. Amonolayer of Cu ions will bind to the His6 tag at the C-terminal ofscFv. The cantilever may then be partially immersed in the scFv solutionfor in-situ monitoring the binding of the scFv to the Cu²⁺ ions on thecantilever tip surface.

Another immobilization method of scFv to a SiO₂ surface entails cleaningan electrode surface using strong acid, reacting the surface withGlycidoxypropyl trimethoxysilane (GOPTS) to generate epoxy groups. Thesurface is then reacted with amine groups of lysine in scFv.Alternatively, it may be possible to utilize other bifunctional linkerssuch as alkoxysilane-PEG-carboxyl on the SiO₂ surface. The surface isthen treated with Cu(ClO₄)˜6H₂O or CuCl₂ to create Cu²⁺ ions on thesurface that to bind the His6 tag or cysteine tag at the c-terminal ofthe scFv. The length of the PEG may vary to obtain optimalantigen-receptor binding.

Example 4

The PEMS of the present invention may also be utilized for simultaneousdetection of multiple antigens using a piezoelectric cantilever array.In an experiment, three piezoelectric cantilever sensors were utilizedfor simultaneous detection of three proteins. One sensor was coated withantibodies specific to PSA, a second sensor was coated with scFvspecific to HER2 and a third was coated with antibodies specific togp120. FIGS. 13( a), 13(b) and 13(c) show the resonance frequency shiftsof three PECS over time in various solutions, one coated with scFv ofHER2, one coated with an antibody to PSA and one coated with an antibodyof gp120. FIG. 13( a) shows the detection results when the PECS areplaced in a HER2 solution of 23 ug/ml. FIG. 13( b) shows the detectionresults when the PECS are placed in a PSA solution of 18 μg/ml, and FIG.13( c) shows the detection results when the PECS are placed in a gp120solution of 100 μg/ml. FIG. 13( a) shows that only the PECS coated withscFv of HER2 responded to the HER2 solution. Similarly, only the PECScoated with antibodies to PSA responded to the PSA solution of FIG. 13(b), and only the PECS coated with antibodies to gp120 responded to thegp120 solution.

The experiment demonstrated that only sensors coated with antibodiesspecific to the antigen solution reacted in each of the three solutions.The experiment proves that a cantilever array can perform in-situ,rapid, simultaneous detection of multiple antigens. A portable devicemay therefore be used to carry multiple sensors for multiple antigendetection in a solution.

Example 5

A number of methods and electrically insulating materials may be used toinsulate the electrodes of a PEMS. Parylene and MTMS, in particular,were separately coated on PZT PEMS and evaluated for dampeningimpedance.

FIG. 14( a) shows the impedance resonance spectrum of a paryleneinsulated PMN—PT/Cu PEMS completely immersed in a static and 1 ml/minflow solution of PBS. The PEMS is insulated with a 1.5 μm thick parylenecoating and has a mass detection sensitivity of about 4×10⁻¹³ g/Hz. Thebackground resonance frequency shift was no more than 30 (60) Hz in 30min in static PBS and in flowing PBS at 1 ml/min. It is expected that ata typical detection flow rate of 0.2-0.5 ml/min, the backgroundresonance frequency shift should be no more than 40 Hz in 30 min. Notethat even at 1 ml/min., the resonance peak still retained a Q value wellover 35.

FIG. 14 (b) compares the peak position shift with time in static PBS andin a 1 ml/min flow. As can be seen the peak positions were fairly stablewith no more than a 30 Hz shift over 30 minutes in static PBS and nomore than a 60 Hz shift over 30 minutes at a flow rate of 1 ml/min inPBS. At the typical flow rate 0.2-0.5 ml/min, the background resonancefrequency shift is expected to be no more than 40 Hz.

FIG. 15( a) shows the resonance spectrum of a PZT PEMS insulated with amonolayer of MTMS. The PEMS was placed in a flow cell and completelyimmersed in a 0.5 ml/min flow solution of PBS. It was found to have amass detection sensitivity of about 5×10⁻¹¹ g/Hz. FIG. 15( b) shows thebackground resonance frequency shift as a function of time in flowingPBS at 0.5 ml/min. The background resonance frequency shift was no morethan 30 Hz over 30 minutes. The graphs show that the electricalimpedance spectrum was stable with strong resonance peak intensity andindicate that the MTMS monolayer is a good electrical insulation layer.

Example 6

A PMN—PT piezoelectric layer or film may be fabricated using aprecursor-suspension method. Submicron crystalline PMN powder wasprepared by dispersing Mg(OH)₂-coated Nb₂O₅ particles in a leadacetate/ethylene glycol solution followed by calcination at about 800°C. The crystalline PMN powder was subsequently suspended in a PTprecursor solution containing lead acetate and titanium isopropoxide inethylene glycol to form a PMN—PT precursor powder, which can be sinteredat a temperature as low as about 900° C.

The PMN—PT films were then polarized in order to obtain highpiezoelectric coefficients. Before polarization, the orientations ofdomains were random with no net polarization. After polarization, manydomains were aligned in the direction of the applied electric fieldresulting in a finite polarization. Although polycrystalline materialsare not easily aligned in comparison to single crystal materials, PMN—PTfilms were geometrically structured so as to facilitate polarizationalignment within the plane of the film.

The resulting freestanding PMN—PT films were then electroplated on oneside with a sputtered 30 nm thick nickel/platinum (Ti/Pt) layer. Acopper layer of appropriate thickness was then electroplated on the Ptsurface to create a non-piezoelectric layer, followed by the depositionof the Ti/Pt electrode on the other face of the film. A PMN—Pt/Cubi-layer was then embedded in wax and cut or chemically etched to form acantilever shape with a wire saw.

A particularly advantageous aspect of this method is the ability toconstruct a microcantilever from one piece of PMN—PT film without havingto separately attach the non-piezoelectric and piezoelectric layers,facilitating the manufacturing process. Because the cantilevers, thusfabricated, had a clean geometry and clean interfaces between the PMN—PTlayer and the electrodes, cantilever sensitivity was heightened, asdemonstrated by Q values as high as 300.

The resulting suspensions have previously been used to formulate PMN—PTfreestanding films of about 8-75 μm thick upon sintering at 1000° C.Typically, the freestanding PMN—PT films have femtogram sensitivity ofat least 2×10⁻¹⁴ g/Hz, dielectric constants greater than 1000, saturatedpolarization of about 30 μC/cm², remnant polarization of 25 μC/cm² and aQ value as high as 300. Therefore PMN—PT microcantilevers are capable ofgenerating higher-mode resonance peaks resulting in enhanced sensitivitydetection.

Example 7

A PZT/SiO₂ piezoelectric layer of film may be formed on silicon wafersand attached to a substrate, such as glass, to form an array. FIG. 3depicts a preferred micro-fabrication procedure for constructingPZT/SiO₂ piezoelectric microcantilever sensors. A low-stress SiO₂substrate, 2-μm in thickness, was first deposited on front and backsides of a (100)-oriented silicon wafer by steam oxidation at 800° C. Afirst side of the SiO₂ constituted the non-piezoelectric layer of themicrocantilever, and a second side of the SiO₂ formed an etching mask,wherein the silicon wafer was wet etched using KOH.

A 400 Å thick TiO₂ bonding layer was then deposited by reactivesputtering of Ti followed by the deposition of a 1500 Å thick platinumelectrode. Reactive sputtering of Ti was carried out under 15 mTorr of80% oxygen and 20% helium. To prepare for deposition of the platinumelectrode, a stage or platform was heated to 650° C. at a rate of 5°C./min. The RF power was adjusted to the expected deposition rate. Afterdeposition, the stage was cooled to room temperature at a rate of 5°C./min to avoid residue stress. Sputtering of the platinum bottomelectrode began when the stage reached room temperature. The resultantplatinum bottom electrode was preferably about 1500 Å in thickness.

A 1.5-μm thick PZT layer was then deposited on the Pt/TiO₂/SiO₂/Sisubstrate using a sol gel method with repeated spin coating and heattreatment steps. According to the sol gel method, titanium isopropoxideand lead acetate were dissolved in ethylene glycol andzirconium-n-propoxide in 2-propanol. The dissolved components were mixedto obtain a PZT precursor solution. The precursor solution alsocontained a 50% excess of lead to compensate for lead loss duringrepeated heat treatment. The PZT thin films were then deposited byrepeated spin coating on a Pt/TiO₂/SiO₂ substrate followed by pyrolysisat 350° C. for 12 min and sintering at 650° C. for 2 hours after every 5depositions. The Pt/TiO₂/SiO₂ substrate was formed by depositing a 2-μmthick low-stress SiO₂ on a silicon wafer through steam oxidation,depositing 40 nm of Ti on the SiO₂ by sputtering and oxidization at 800°C. and depositing 150 nm Pt by sputtering to prevent undesiredinterfacial reaction and diffusion. The TiO₂ buffer and a SiO₂ layer,having a thickness larger than 0.5 μm, together effectively eliminateddiffusion between the PZT layer and the substrate.

After forming the PZT layer, a first electrode, Pt/Ti or Au/Cr, wasdeposited by E-gun evaporation in high vacuum (2×10⁻⁶ torr or lower) toavoid oxidation of metal, particularly oxidation of the Cr layer. E-gunevaporation was used because it is gentler than sputtering or thermalevaporation and avoids damaging the PZT layer. The thickness of theresultant Ti or Cr bonding layer was approximately 5-40 nm and theresultant Pt or Au electrode layer was approximately 100-200 nm thick. Anickel layer was deposited on the top electrode to form a hard mask forprotecting the top electrode and the underlying PZT during dry etching.The Ni and top electrode were patterned using an over-hang lift-offprocess. LOR10B and SPR3012 photoresists are recommended for executingthe over-hang lift-off process, and the thickness of the undercutphotoresist (LOR) was about 0.8 to 1.2 μm, approximately 1.5 timeslarger than the metal layer. The selectivity or etching rate ratio ofPZT to Ni was nearly 5:1. The thickness of the resultant Ni layer wasabout 0.3-0.5 μm, depending on the PZT thickness.

The exposed PZT and TiO₂/Pt bottom electrode was patterned by achlorine-based inductively coupled plasma (ICP) dry etching processusing a chlorine etching gas to expose the SiO₂ and 150 sccm flow rate.The pressure of the etching chamber was set to below 10 mTorr. The RFpower was adjusted to the etching rate. Since the PZT is a ceramicmaterial and the nickel etching mask is stable at high temperatures, ahigh RF power of about 400 W is recommended for PZT dry etching. Theselectivity ratios of the PZT dry etching process were 5:1 for Ni and8:1 for Pt. The etching mask for the bottom electrode was a thickphotoresist such as one in the SPR220 series. Because the etching maskwas a photoresist, a low RF power was recommended for this etchingprocess. Silver glue was used to fill spaces between the sample and thestage to help dissipate heat during etching. For every 5 min of etching,a 20-min cooling period was required. By keeping the etching temperaturebelow 120° C., the PR was easily removed with acetone after etching.

The silicon dioxide on the second side was patterned byphotolithography, and the exposed silicon dioxide was etched by CF₄/CHF₃based reactive ion etching (RIE). After the silicon-dioxide etching isfinished, the whole first side was sealed by black wax, and the samplewas placed in a 45% potassium hydroxide (KOH) solution. The temperatureof the solution was set to 55-60° C., and the KOH was used to etch theexposed silicon with the remaining silicon dioxide as an etching mask.

After etching, the black wax was dissolved using trichloroethylene (TCE)and the front side was cleaned. The exposed silicon dioxide was removedon the front side by a CF₄/CHF₃-based reactive ion etching (RIE). Thefirst electrode and second electrode work together because the mask andthe selectivity ratios are very high. If desired, the etching durationmay be increased to ensure that all the silicon dioxide is removed andthe cantilevers are released. After rinsing with acetone, 2-proponal anddeionized water, free-standing PZT/SiO₂ piezoelectric microcantileversensors were obtained.

Example 8

Mercaptopropylsilane (MPTS) was investigated for its ability to functionboth as an electrical insulator and as receptor immobilizer. The resultsestablished that MPTS is an effective insulator and immobilizer enablingthe detection of the HER2 antibody both in solution and in the presenceof BSA.

To prepare the MPTS coated PEMS, a PMN—PT/tin PEMS was first soaked in adiluted (1:100 in water) piranha solution, containing two parts 98%sulfuric acid and one part of 30% hydrogen peroxide, at 20° C. for 1 minto clean the tin and gold surfaces. Next, the PEMS was submerged in abeaker containing a 40 mM solution of MPTS in ethanol, and the beakerwas covered with paraffin film to prevent ethanol evaporation. After 4hours, the PEMS was rinsed with DI water and submerged in a 0.01 Msolution of NaOH overnight for cross-linking. The PEMS was then soakedin DI water for 1 hour and dried overnight in a vacuum-oven at 762 mm Hgto ensure all the water was removed. The PEMS was then submerged in a 1volume percent solution of MPTS in ethanol titrated to a pH 4.5 withacetic acid. The solution was covered with paraffin film to preventethanol evaporation, and the PEMS was allowed to soak for 8 hours. ThePEMS was again soaked in DI water for 1 hour and dried overnight in avacuum-oven at 762 mm Hg.

To immobilize the scFv receptors on the PEMS surface,sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(Sulfo-SMCC) was first mixed with scFv to form a peptide bond between aprimary amine of the scFv and the NHS ester of the Sulfo-SMCC asdepicted in FIG. 16( a). To effectively bind SMCC to scFv, 5 mM SMCC wasmixed with 400 nM scFv for 2 hrs. The unreacted SMCC was then removed byrepeated microcentrifugation with a 10 kD filter a total of 4 times. TheMPTS-coated PEMS was then soaked in the scFv-bound bound SMCC solution.

FIG. 16( b) illustrates the reaction between the sulfhydryl of the MPTSon the sensor surface with the maleimide of the scFv-bound SMCC toimmobilize the scFv. The immobilized scFv has random orientation. Afterimmobilization, the PEMS was placed in a mild quenching solution of DIwater for 1 hr to quench the unreacted NHS functionalities on the SMCC.Since any primary amine of the scFv could react with the NHS ester ofthe SMCC the resultant immobilized scFv would be randomly oriented asschematically shown in FIG. 16( b). With the cysteine tag engineered atthe base of the scFv, it is now possible to covalently bind the scFv tothe sensor surface and at the same time orient the scFv for opticalantigen-scFv binding.

For HER2 detection, the scFv-immobilized PMN—PT/tin PEMS was immersed ina home-built flow cell with a peristaltic pump. First PBS was allowed toflow across the conjugated surface for 10 minutes in order to obtain abackground reading. Next, a blocking solution of 10 mg/ml of BSA in PBSwas allowed to flow over the surface of the cantilever until nosignificant deviations are observed in the cantilevers resonantfrequency. The cantilever was then gently rinsed with a solution ofTWEEN-20 followed by PBS. PBS was then allowed to flow across thesurface of the cantilever for 10 minutes to ensure that there was nobackground drift. The flow cell contained 3 ml of HER2 solution. Thedetection was carried out with two faces of the PEMS tangential to theflow at a flow rate of 0.5 ml/min. After each detection, the HER2 wasreleased from the PEMS surface by flushing with a glycine/HCl solutionhaving a pH of about 2.5 or a supersaturated solution of NaCl. Followingthe release, the PEMS was then exposed to a suspension of a differentconcentration for another detection run.

FIGS. 17( a)-17(c) show the in-situ results of scFv immobilization, thesubsequent BSA blocking and detection of 100 ng/ml of HER2 alone, and inthe background environment of BSA, using the MPTS-insulated PMN—PT/SnPEMS. FIG. 17( b) shows that the MPTS-coating was effective as anelectrical insulation layer as the resonance peak maintained the samehigh Q value throughout the 2 hour detection period. In addition, FIGS.17( a) and 17(c) illustrate that HER2 can be detected in the backgroundenvironment of BSA. After BSA blocking, the resonance frequency shiftfor detecting HER2 alone, FIG. 17( a), and in the presence of BSA, (FIG.17( c), was roughly the same, indicating that the BSA blocking procedureeffectively saturated the nonspecific binding sites of BSA and had noobservable effect to the PEMS resonance frequency shift. FIG. 18 showsthe resonance frequency shift versus time in HER2 detection at 5, 10,and 50 ng/ml in a background of 1 mg/ml BSA using the MPTS-insulatedPMN—PT/Sn PEMS. As can be seen, the detection yielded −50 Hz, −150 Hz,and −700 Hz after 50 min at 5, 10, and 50 ng/ml, respectively,indicating that the present PMN—PT/Sn PEMS is capable of detecting HER2in a background of BSA whose concentration was one million fold higherthan that of HER2.

Example 9

The PEMS is capable of in situ detection applications. A one-sided PEMSwas able to detect the presence of HER2 in the presence of Bovine SerumAlbumin (BSA).

The tin surface of a PMN—PT/Sn PEMS was insulated using MTMS. The scFvreceptors were immobilized only on the platinum side of the electrode byfirst coating the platinum surface with an activated MPA self assemblymonolayer to bind to a primary amine of the scFv.

A one sided PMN—PT/tin PEMS was found capable of detecting of HER2 at aconcentration of 1 ng/ml in the presence of 1 mg/ml of BSA in a 30 mlsolution flowing at a rate of 1 ml/min. FIG. 19 shows a resonanceincrease in the presence of HER2, indicating that the PEMS was capableof detecting 1 ng/ml of HER2 in 1 mg/ml of BSA.

The BSA did not adhere to the MPA surface to the same degree as comparedto the MPTS—SMCC surface of FIGS. 17( a) and 17(c). In the presence of 1mg/ml BSA, the resonance frequency of the PMN—PT/tin PEMS was stable asshown at t=150 of FIG. 19. In comparison, in the presence of 1 ng/ml ofHER2 and 1 mg/ml of BSA, the resonance frequency of the PEMS increasedby about 200 Hz during a 160<t<240 interval. Upon the removal of theHER2, the resonance frequency stabilized, suggesting that the one-sidedPMN—PT/tin was able to detect the presence of HER2 at 1 ng/ml in thepresence of 1 mg/ml of BSA. Note that resonance frequency increase uponbinding of target antigens on the sensor surface is common withone-sided detection due to the stress imbalance from the one-sidedbinding.

FIGS. 17-19 clearly demonstrate that PMN—PT/tin PEMS were able to detectHER2 in situ at a concentration as low as 1 ng/ml in the presence of a 1mg/ml BSA background, which is lower than the normal 15 ng/ml HER2concentration of a typical healthy woman. Therefore, these resultssuggest that PEMS would be able to detect breast cancer markers in serumwith useful levels of sensitivity and specificity.

Example 10

A 900 μm long and 500 μm wide PMN—PT/Cu PEMS having an 8 μm thick PMN—PTlayer and a 3 μm thick copper layer was used to detect the presence ofHER2 in diluted fetal bovine serum (FBS).

The PEMS was prepared using mercaptopropyltrimethoxysilane (MPS) as aninsulation layer and covalent receptor conjugation. Biotin wascovalently bound to the sulfhydryl of the MPS usingmaleimide-PEO₂-biotin (Pierce). Avidin was subsequently attached to thebiotin by soaking the surface of the cantilever in a 1 mg/ml solution ofneutravidin (Pierce). Sulfosuccinimidyl-6-[biotinamido]-6-hexanamidohexanoate (Pierce) was used to attach biotin to the primary amine in ascFv. The avidin coated PEMS was then submerged in the solutioncontaining the biotinylated scFv for 60 minutes at 4° C. The cantileverwas subsequently soaked in a 3% BSA solution for 2 hours to facilitateblocking of unreacted areas. The PEMS was then rinsed with a 1% BSAsolution and a 1% Tween® 20 solution.

For HER2 detection, the scFv-immobilized PEMS was then immersed in aflow cell containing about 6 ml of liquid with a peristaltic pump. Afterconjugation, the PEMS was exposed to a solution containing diluted 1:40bovine serum and HER2 in one of the following concentrations: about 110pg/ml, 50 pg/ml, 30 pg/ml, and 10 pg/ml. FIG. 20 shows the resonancefrequency shift versus time for the PEMS. As can be seen, the PEMSyielded a resonance frequency shift of about 2800±200, 1000±100,140±100, and 10±100 Hz at t=50 minutes, for 110, 50, 30 and 10 ng/mlHER2 concentrations, respectively. The results indicate the PEMS wasable to detect breast cancer markers in diluted serum with aconcentration limit of 30 pg/ml.

Example 11

An array of three millimeter long PZT/glass PEMS were used to assess thebinding ability of a receptor for its ligand. The performance of 3receptors, B1 scFv, H3 scFv, and Herceptin, was compared using an arrayof three identical PZT/glass PEMS insulated with MPS. Aheterobifunctional cross linking agent, sulfosuccinimidyl4-N-maleimidomethyl cyclohexane-1-carboxylate, was used to covalentlyattach the receptor to the MPS surface. After receptor immobilizationthe cantilevers were blocked using a 3% BSA solution for 1 hour, andthen rinsed with a 1% BSA solution and a 1% Tween® 20 solution.

The three PEMS were all completely submerged in the same 6 ml flow cell.A 1:40 fetal bovine serum diluted in PBS containing a known spikedamount of HER2 ECD was introduced into the flow cell. The sample wasflowed through the system at a rate of 0.7 ml/minute. FIG. 21 shows theresultant PEMS detection of HER2 in solutions containing diluted 1:40fetal bovine serum and HER2 in concentrations of 6 ng/ml, 60 ng/ml or600 ng/ml. In Table 3, the resonance frequency shift is given at t=60minutes. According to FIG. 21 and Table 3, the three identical PEMSshowed that Herceptin consistently had larger frequency shifts,suggesting that Herceptin can be detected using PEMS at a lowerconcentration limit, as compared to B1 scFv and H3 scFv.

TABLE 3 Resonance frequency shift of the array PZT/glass PEMSimmobilized with different HER2 receptor: H3, B1 and Herceptin.Concentration H3 B1 Herceptin  6 ng/ml 55 460 850  60 ng/ml 150 840 1500600 ng/ml 780 1300 3200

Example 12

An array of millimeter long PZT/glass PEMS, each having a width of about300-600 μm and including a 1 mm long PZT layer and a 2 mm long glasstip, as shown in FIG. 22( a), was used to detect the water borneparasite, cryptosporidium parvum (CP) at concentration levels of 0.1CP/ml in a volume of 60 ml, i.e., 6 total CPs in the 60 ml of water. ThePEMS were insulated with MPS and conjugated with the anti-CP IgM usingthe bi-functional linker SMCC.

In the experiment, two arrays were used; one control array, whose PEMSwere coated with an irrelevant protein BSA (not shown), served ascontrol and one experimental array, whose PEMS were coated with anti-CPIgM. A CP sample was introduced to the PEMS array at a flow rate ofabout 1 ml/min. FIG. 22( b) shows the resonance frequency shift as afunction of time for each of the four experimental PEMS coated withanti-CP IgM, i.e. C1, C2, C3, and C4. As can be seen, the control arrayshowed no resonance frequency shift while C1, C2, and C3 showed severalhundred Hz resonance frequency shifts, indicating that a PEMS array maybe used for CP detection at very low concentrations based on thepositive response from any of the PEMS, which indicates the presence ofa target pathogen.

Example 13

A 40 μm long PZT/SiO₂ PEMS, as shown in FIG. 23( a), was found to havean improved sensitivity of about 10⁻¹⁶ g/Hz with resonance peak Q valuesas high as 300 in a humidity controlled environment. FIG. 23( b) showsthe resonance frequency spectra of the PEMS corresponding to varyinghumidity. The resonance frequency was found to decrease with anincreasing relative humidity due to the adsorption of water molecules onthe sensor surface. Therefore it is possible to increase sensitivitywith very small PEMS.

The resultant sensitivity of 6×10⁻¹⁶ g/Hz demonstrates the feasibilityof further reducing PEMS size while increasing sensitivity when comparedto the millimeter sized commercial PZT PEMS having a sensitivity ofabout 10⁻¹¹ g/Hz and 300-800 μm long PMN—PT PEMS having a sensitivity ofabout 10⁻¹³ to 10⁻¹⁴ g/Hz.

Having described the preferred embodiments of the invention which areintended to be illustrative and not limiting, it is noted thatmodifications and variations can be made by persons skilled in the artin light of the above teachings. It is therefore to be understood thatchanges may be made in the particular embodiments of the inventiondisclosed which are within the scope and spirit of the invention asoutlined by the appended claims. Having thus described the inventionwith the details and particularity required by the patent laws, theintended scope of protection is set forth in the appended claims.

The invention claimed is:
 1. A method for using a piezoelectricmicrocantilever sensor comprising the steps of: actuating amicrocantilever sensor having a Q value greater than 20, comprising apiezoelectric layer, a non-piezoelectric layer, and a receptor, whereinsaid piezoelectric layer has a thickness of about 1 μm to about 127 μm,a dielectric constant of at least 1600, and a piezoelectric coefficient−d₃₁ of at least about 250 pm/V; binding a molecule or compound to saidreceptor, detecting a force exerted on said piezoelectric layer by saidbound molecule or compound, and determining one or more of a presence ofsaid molecule or compound and a concentration of said molecule orcompound from said detected force.
 2. The method of claim 1, whereinsaid receptor is selected from the group consisting of DNA, proteins,enzymes, cells, viruses, parasites, antigens and pathogens.
 3. Themethod of claim 1, wherein said molecule or compound is selected fromthe group consisting of bioterrorism agents, cancer agents, bacterialdisease agents and viral disease agents.
 4. The method of claim 1,further comprising the step of detecting a resonance frequency shift. 5.The method of claim 1, wherein the microcantilever sensor furthercomprises an electrical insulation layer which insulates conductingelements of said sensor.
 6. The method of claim 5, wherein theelectrical insulation layer comprises a material selected from the groupconsisting of poly-para-xylylene, methyltrimethoxysilane,3-mercaptopropyl trimethoxysilane, Al₂O₃, SiO₂ and functionalizedhydrophobic silanes and mixtures thereof.
 7. The method of claim 1,wherein the receptor is bound to a conducting element by animmobilization layer.
 8. The method of claim 1, wherein a length of saidpiezoelectric layer is less than or greater than a length of saidnon-piezoelectric layer.
 9. The method of claim 1, wherein saidpiezoelectric layer if fabricated from a freestanding film.
 10. Themethod of claim 1, wherein said piezoelectric layer has a thickness ofless than 2 μm.
 11. The method of claim 1, wherein said piezoelectriclayer has a dielectric constant of at least 1900 and a thickness lessthan 4 μm.
 12. The method of claim 1, wherein said piezoelectric layerhas a dielectric constant of at least 1900 and a thickness less than 2μm.
 13. The method of claim 1, wherein said piezoelectric layer has athickness less than 8 μm.
 14. The method of claim 1, wherein saidpiezoelectric layer comprises a material selected from the groupconsisting of lead magnesium niobate-lead titanate,lead-zirconate-titanate, and doped sodium potassium niobate-lithiumniobate.
 15. The method of claim 1, wherein said piezoelectric layercomprises a lead-free piezoelectric material.
 16. The method of claim 1,wherein said piezoelectric microcantilever has a Q value greater than120.
 17. The method of claim 1, wherein the non-piezoelectric layercomprises a material selected from the group consisting of a ceramicmaterial, a polymeric material, a metallic material and a combinationsthereof.
 18. The method of claim 1, wherein said non-piezoelectric layercomprises a material selected from the group consisting of silicondioxide, silicon nitride, tin and copper.
 19. The method of claim 1,wherein said non-piezoelectric layer comprises multiple layers ofdifferent materials.
 20. The method of claim 1, wherein the step ofactuating comprises applying a voltage across said piezoelectric layer.